Targeted introduction of DNA into primary or secondary cells and their use for gene therapy

ABSTRACT

The present invention relates to a method of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. That is, it relates to a method of introducing DNA into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site. The present invention further relates to primary or secondary cells, referred to as homologously recombinant (HR) primary or secondary cells, produced by the present method and to uses of the homologously recombinant primary or secondary cells. The present invention also relates to a method of turning on a gene present in primary cells, secondary cells or immortalized cells of vertebrate origin, which is normally not expressed in the cells or is not expressed at significant levels in the cells.

This application is a continuation of application Ser. No. 07/789,188filed Nov. 5, 1991, abandoned.

BACKGROUND OF THE INVENTION

The ability to move DNA from one cell to another is a powerful tool inmodern molecular biology, yet the idea that this movement might bepossible predates the current revolution in genetic engineering. In1928, Griffith paved the way for the discovery that nucleic acids arethe genetic material when he noticed that the virulence of bacteriacould be altered by mixing live bacteria with solutions derived fromkilled bacteria. By the early 1960's, not only was the structure of therelevant component of the solution, DNA, solved, but it was alreadyestablished that DNA could be moved into mammalian cells (Syzbalski,1961). The focus of these early days of molecular biology and tissueculture were irreversibly changed by two critical developments: thediscovery of calcium phosphate precipitation, a simple procedure tointroduce DNA into immortalized cells in culture (Graham and van der Eb,1972) and the isolation and characterization of mammalian globin,insulin, and growth hormone genes in the mid- to-late 1970's.

Today, the ability to manipulate DNA and to introduce it into cells hasprofound practical implications for human health. Recombinant proteinsproduced by such manipulations are becoming widely accepted treatmentsfor a number of human diseases and play major roles in agriculture.Though far less developed, the field of human gene therapy also has beenand will continue to be influenced by improvements in technologies forthe manipulation of DNA.

Gene therapy is a medical intervention in which a small number of thepatient's cells are modified genetically to treat or cure any condition,regardless of etiology, that will be ameliorated by the long-termdelivery of a therapeutic protein. Gene therapy can, therefore, bethought of as an in vivo protein production and delivery system, andalmost all diseases that are currently treated by the administration ofproteins (as well as several diseases for which no treatment iscurrently available), are candidates for treatment using gene therapy.The field can be divided into two areas: germ cell and somatic cell genetherapy. Germ cell gene therapy refers to the modification of spermcells, egg cells, zygotes or early stage embryos. On the basis of bothethical and practical criteria, germ cell gene therapy is inappropriatefor human use. From an ethical perspective, modifying the germ linewould change not only the patient, but also the patient's offspring and,to a small but significant extent, the human gene pool as a whole.

In contrast to germ cell gene therapy, somatic cell gene therapy wouldaffect only the person under treatment (somatic cells are cells that arenot capable of developing into whole individuals and include all of thebody's cells with the exception of the germ cells). As such, somaticcell gene therapy is a reasonable approach to the treatment and cure ofcertain disorders in human beings. In a somatic cell gene therapysystem, somatic cells (i.e., fibroblasts, hepatocytes or endothelialcells) are removed from the patient, the cells are cultured in vitro,the gene(s) of therapeutic interest are added to the cells and thegenetically-engineered cells are characterized and reintroduced into thepatient. The means by which these five steps are carried out are thedistinguishing features of a given gene therapy system.

To provide an overview of how somatic cell gene therapy might be appliedin practice, an example concerning the treatment of hemophilia B will beconsidered. Hemophilia B is a bleeding disorder that is caused by adeficiency in Factor IX, a protein normally found in the blood. As acandidate for a gene therapy cure, an affected patient would have anappropriate tissue removed (i.e., bone marrow biopsy to recoverhematopoietic stem cells, phlebotomy to obtain peripheral leukocytes, aliver biopsy to obtain hepatocytes or a punch biopsy to obtainfibroblasts or keratinocytes). The patient's cells would be isolated,genetically engineered to contain an additional Factor IX gene thatdirects production of the missing Factor IX and reintroduced into thepatient. The patient is now capable of producing his or her own FactorIX and is no longer a Hemophiliac. The physician will most likelyschedule close follow up in the weeks and months after the treatment,but in a literal sense, the patient would have been cured.

In state-of-the-art somatic cell gene therapy systems, it is notpossible to direct or target the additional therapeutic DNA to apreselected site in the genome. In fact, in retrovirus-mediated genetherapy, the most widely utilized experimental system retrovirusesintegrate randomly into independent chromosomal sites in millions tobillions of cells. This mixture of infected cells is problematic in twosenses: first, since integration site plays a role in the function ofthe therapeutic DNA, each cell has a different level of function and,second, since the integration of DNA into the genome can triggerundesired events such as the generation of tumorigenic cells, thelikelihood of such events is dramatically increased when millions tobillions of independent integrations occur.

The problems of populations consisting of large numbers of independentintegrants might be avoided in two ways. First, a single cell with arandom integration site could be propagated until sufficient numbers ofthe cloned cell could be introduced into the individual. The cells thatmake up this clonal population would all function identically. Inaddition, only a single integration site would be present in the clonalpopulation, significantly reducing the possibility of a deleteriousevent. Second, a single cell or a population of cells could be treatedwith therapeutic DNA such that the DNA sequences integrate into apreselected site in the genome. In this case, all the cells would beengineered identically and function identically. Furthermore, the riskof a deleterious integration event would be eliminated. Both the abovesolutions are demonstrated in this application.

The application of targeting to somatic cell gene therapy has severalother advantages in addition to simply introducing additional genes orfunctional DNA sequences into a cell. In targeted gene therapy, it wouldbe possible to repair, alter, replace or delete DNA sequences within thecell. In the illustration of somatic cell gene therapy discussed above,for example, targeting would allow the patient's non-functional FactorIX gene to be repaired. The ability to repair, alter, replace and deleteDNA sequences utilizing targeting technology would expand the range ofdiseases suitable for treatment using gene therapy (and for the in vitroproduction of recombinant proteins as well). As the above discussionsuggests, it would be extremely useful to be able to target primary andsecondary vertebrate cells.

SUMMARY OF THE INVENTION

The present invention relates to a method of gene or DNA targeting incells of vertebrate, particularly mammalian, origin. That is, it relatesto a method of introducing DNA into primary or secondary cells ofvertebrate origin through homologous recombination or targeting of theDNA, which is introduced into genomic DNA of the primary or secondarycells at a preselected site. The preselected site determines thetargeting sequences used. The present invention further relates tohomologously recombinant primary or secondary cells, referred to ashomologously recombinant (HR) primary or secondary cells, produced bythe present method and to uses of the HR primary or secondary cells. Thepresent invention also relates to a method of turning on a gene presentin primary cells, secondary cells or immortalized cells of vertebrateorigin, which is normally not expressed in the cells or is not expressedat significant levels in the cells. Homologous recombination ortargeting is used to replace the regulatory region normally associatedwith the gene with a regulatory sequence which causes the gene to beexpressed at significant levels in the cell.

As described herein, Applicants have demonstrated gene or DNA targetingin primary and secondary cells of mammalian origin. Prior to the presentwork, gene targeting had been reported only for immortalized tissueculture cell lines (Mansour, Nature 336:348-352 (1988); Shesely, PNAS88:4294-4298 (1991); Capecchi, M. R., Trends in Genetics 5:70-76(1989)). As a result of the work described herein, it is now possible tostably integrate exogenous DNA into genomic DNA of a host or recipientprimary or secondary cell. The exogenous DNA either encodes a product,such as a therapeutic protein or RNA, to be expressed in primary orsecondary cells or is itself a therapeutic product or other productwhose function in primary or secondary cells is desired.

As used herein, the term primary cell includes cells present in asuspension of cells isolated from a vertebrate tissue source (prior totheir being plated, i.e., attached to a tissue culture substrate such asa dish or flask), cells present in an explant derived from tissue, bothof the previous types of cells plated for the first time, and cellsuspensions derived from these plated cells. The term secondary cell orcell strain refers to cells at all subsequent steps in culturing. Thatis, the first time a plated primary cell is removed from the culturesubstrate and replated (passaged), it is referred to herein as asecondary cell, as are all cells in subsequent passages. Secondary cellsare cell strains which consist of secondary cells which have beenpassaged one or more times. A cell strain consists of secondary cellsthat: 1) have been passaged one or more times; 2) exhibit a finitenumber of mean population doublings in culture; 3) exhibit theproperties of contact-inhibited, anchorage dependent growth(anchorage-dependence does not apply to cells that are propagated insuspension culture); and 4) are not immortalized. A "clonal cell strain"is defined as a cell strain that is derived from a single founder cell.A "heterogenous cell strain" is defined as a cell strain that is derivedfrom two or more founder cells.

In the method of the present invention, cells to be transfected withexogenous DNA are combined with a DNA construct comprising the exogenousDNA, targeting DNA sequences and, optionally, DNA encoding one or moreselectable markers and the resulting combination is treated in such amanner that the DNA construct enters the cells. This is accomplished bysubjecting the combination to electroporation, microinjection, or othermethod of introducing DNA into vertebrate cells (e.g., calcium phosphateprecipitation, modified calcium phosphate precipitation, microprojectilebombardment, fusion methodologies, receptor mediated transfer, orpolybrene precipitation). Once in the cell, the exogenous DNA isintegrated into the cell's genomic DNA by homologous recombinationbetween DNA sequences in the DNA construct and DNA sequences in thegenomic DNA. The sequences involved in targeting (i.e., those whichparticipate in homologous recombination with genomic sequences) can bepart of the exogenous DNA or can be separate from (in addition to) theexogenous DNA. The result is homologously recombinant (HR) primary orsecondary cells in which the exogenous DNA, as well as other DNAsequences present in the DNA construct, are stably integrated intogenomic DNA.

The present method of targeting exogenous DNA has a wide variety ofapplications. These applications fall into three general types orcategories: 1) addition of DNA to sequences already present invertebrate cells; 2) replacement of DNA sequences present in vertebratecells; and 3) deletion of sequences normally present in vertebratecells. For example, the present method can be used to modify primary orsecondary cells in order to repair, alter, delete or replace a resident(host cell) gene; to introduce a gene encoding a therapeutic or otherproduct not expressed at significant levels in the primary or secondarycells as obtained; to introduce regulatory sequences into primary orsecondary cells; to repair, alter, delete or replace regulatorysequences present in primary or secondary cells; to knock out(inactivate) or remove an entire gene or a gene portion; to produceuniversal donor cells (e.g., by knocking out cell surface antigens), andto augment production of a gene product already made in the HR primaryor secondary cell.

The present method is particularly useful for producing homologouslyrecombinant cells to be used for in vivo protein production anddelivery, as described in commonly owned U.S. patent applicationentitled "In Vivo Protein Production and Delivery System for GeneTherapy", U.S. Ser. No. 07/787,840, filed of even date herewith. Theteachings of the patent application entitled "In Vivo Protein Productionand Delivery System for Gene Therapy" are incorporated herein byreference.

The present method of targeting is particularly useful to turn on a genewhich is present in a cell (primary, secondary or immortalized) but isnot expressed in or is not expressed at significant levels in the cellsas obtained. The present method can be used for protein production invitro or for gene therapy. For example, it can be used to turn on genes,such as the human erythropoietin, growth hormone and insulin genes andother genes (e.g., genes encoding Factor VIII, Factor IX,erythropoietin, alpha-1 antitrypsin, calcitonin, glucocerebrosidase,growth hormone, low density lipoprotein (LDL) receptor, IL-2 receptorand its antagonists, insulin, globin, immunoglobulins, catalyticantibodies, the interleukins, insulin-like growth factors, superoxidedismutase, immune response modifiers, parathyroid hormone, interferons,nerve growth factors, tissue plasminogen activators, and colonystimulating factors) in a cell of any type (primary, secondary orimmortalized). In this embodiment, a gene's existing regulatory regioncan be replaced with a regulatory sequence (from a different gene or anovel regulatory sequence made by genetic engineering techniques) whosepresence in the cell results in expression of the gene. Such regulatorysequences may be comprised of promoters, enhancers, Scaffold-attachmentregions, negative regulatory elements, transcriptional initiation sites,regulatory protein binding sites or combinations of these sequences. Asa result, an endogenous copy of a gene encoding a desired gene productis turned on (expressed) and an exogenous copy of the gene need not beintroduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of plasmid pXGH5, which includesthe human growth hormone (hGH) gene under the control of the mousemetallothionein promoter.

FIG. 2 is a schematic representation of plasmid pcDNEO, which includesthe neo coding region (BamHI-BglII fragment) from plasmid pSV2neoinserted into the BamHI site of plasmid pcD; the Amp-R and pBR322orisequences from pBR322; and the polyA, 16S splice junctions and earlypromoter regions from SV40.

FIG. 3 is a schematic representation of plasmid pXGH301 which includesthe human growth hormone gene and the neo gene.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, Applicants have demonstrated that DNA can beintroduced into primary or secondary vertebrate cells in a DNA constructor plasmid and integrated into the genome of the transfected primary orsecondary cells by homologous recombination. That is, they havedemonstrated gene targeting in primary and secondary mammalian cells.They have further demonstrated that the exogenous DNA has the desiredfunction in the homologously recombinant (HR) cells and that correctlytargeted cells can be identified on the basis of a detectable phenotypeconferred by a selectable marker gene.

Applicants describe (Example 1) construction of plasmids containing aselectable marker gene (plasmid pcDNEO), a gene encoding a therapeuticproduct (plasmid pXGH5) or both (pXGH301). They also describeconstruction of a plasmid useful for targeting to a particular locus(the HPRT locus) in the human genome and selection based upon a drugresistant phenotype (Example 2). This plasmid is designated pE3NEO andits integration into the cellular genomes at the HPRT locus producescells which have an hprt⁻, 6-TG resistant phenotype and are also G418resistant. As described, they have shown that pE3NEO functions properlyin gene targeting in an established human fibroblast cell line (Example3), by demonstrating localization of the DNA introduced into establishedcells within exon 3 of the HPRT gene.

In addition, Applicants demonstrate gene targeting in primary andsecondary human skin fibroblasts using pE3Neo (Example 4) and describeconstruction of a plasmid for targeted insertion of a gene encoding atherapeutic product (human growth hormone [hGH]) into the human genome(Example 5). The subject application further teaches modification of DNAtermini to enhance targeting of DNA into genomic DNA (Example 6) andconstruction of a variety of targeting plasmids. For instance,Applicants describe targeting plasmids for placing a human gene underthe control of a murine promoter known to function in human cells(Examples 7 and 10); for targeting to sequences flanking a gene andisolation of targeted secondary fibroblasts using a variety of screeningand selection approaches (Examples 8, 9, 11 and 12); for placing a humangene not normally expressed in the primary or secondary cells under thecontrol of a promoter of nonhuman or human origin, to produce HR primaryor secondary cells which express the encoded product (Examples 7-12).

Using the methods and DNA constructs or plasmids taught herein ormodifications thereof which are apparent to one of ordinary skill in theart, exogenous DNA which encodes a therapeutic product (e.g., protein,ribozyme, nucleic acid) can be inserted at preselected sites in thegenome of vertebrate (e.g., mammalian, both human and nonhuman) primaryor secondary cells.

The methods and DNA constructs described can be used for a wide varietyof purposes. The method can be used to alter primary or secondary cellsof vertebrate origin in order to repair, alter, delete or replace DNAalready present in the recipient primary or secondary cell; to introduceinto primary or secondary cells a gene or DNA sequence (at a preselectedsite) which encodes a therapeutic product or other desired product or isitself a therapeutic or other product; to add to or replace regulatorysequences present in the primary or secondary cell recipients; to knockout or remove an entire gene or gene portion present in primary orsecondary cells; and to produce universal donor cells.

Transfected Cells

Primary and secondary cells to be transfected by the present method canbe obtained from a variety of tissues and include all cell types whichcan be maintained in culture. For example, primary and secondary cellswhich can be transfected by the present method include fibroblasts,keratinocytes, epithelial cells (e.g., mammary epithelial cells,intestinal epithelial cells), endothelial cells, glial cells, neuralcells, formed elements of the blood (e.g., lymphocytes, bone marrowcells), muscle cells, hepatocytes and precursors of these somatic celltypes. Primary cells are preferably obtained from the individual to whomthe transfected primary or secondary cells are administered. However,primary cells may be obtained from a donor (other than the recipient) ofthe same species or another species (e.g., nonhuman primates, mouse,rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Transfected primary and secondary cells have been produced, with orwithout phenotypic selection, as described in co-pending applicationentitled "In Vivo Protein Production and Delivery System for GeneTherapy" (Attorney's Docket No. TKT91-01), filed of even date herewithand shown to express exogenous DNA encoding a therapeutic product.

Exogenous DNA

Exogenous DNA incorporated into primary or secondary cells by thepresent method is: 1) DNA which encodes a translation or transcriptionproduct whose expression in primary or secondary cells is desired, suchas a product useful to treat an existing condition or prevent it fromoccurring and 2) DNA which does not encode a gene product but is itselfuseful, such as in treating an existing condition or preventing it fromoccurring.

DNA incorporated into primary or secondary cells can be an entire geneencoding an entire desired product or a gene portion which encodes, forexample, the active or functional portion(s) of the product. The productcan be, for example, a hormone, a cytokine, an antigen, an antibody, anenzyme, a clotting factor, a transport protein, a receptor, a regulatoryprotein, a structural protein, an anti-sense RNA, a ribozyme or aprotein or a nucleic acid which does not occur in nature (i.e., a novelprotein or novel nucleic acid). The DNA can be obtained from a source inwhich it occurs in nature or can be produced, using genetic engineeringtechniques or synthetic processes. The DNA transfected into primary orsecondary cells can encode one or more therapeutic products. Aftertransfection into primary or secondary cells, the exogenous DNA isstably incorporated into the recipient cell's genome (along with theadditional sequences present in the DNA construct used), from which itis expressed or otherwise functions.

Selectable Markers

A variety of selectable markers can be incorporated into primary orsecondary cells. For example, a selectable marker which confers aselectable phenotype such as drug resistance, nutritional auxotrophy,resistance to a cytotoxic agent or expression of a surface protein, canbe used. Selectable marker genes which can be used include neo, gpt,dhfr, ada, pac, hyg, mdrl and hisD. The selectable phenotype conferredmakes it possible to identify and isolate recipient primary or secondarycells. Selectable markers can be divided into two categories: positiveselectable and negative selectable. In positive selection, cellsexpressing the positive selectable marker are capable of survivingtreatment with a selective agent (such as neo, gpt, dhfr, ada, pac, hyg,mdrl and hisD). In negative selection, cells expressing the negativeselectable marker are destroyed in the presence of the selective agent(e.g., tk, gpt).

DNA Constructs

DNA constructs, which include exogenous DNA encoding a desired product,targeting sequences for homologous recombination and, optionally, DNAencoding one or more selectable markers are used to transfect primary orsecondary cells in which homologous recombination is to occur. In thisembodiment, DNA sequences necessary for expression of the exogenous DNAwill generally be present as well. DNA constructs which includeexogenous DNA sequences which do not encode a gene product (and are thedesired product) and, optionally, include DNA encoding a selectablemarker, can also be used to transfect primary and secondary cells.

The exogenous DNA, targeting sequences and selectable marker can beintroduced into cells on a single DNA construct or on separateconstructs. The total length of the DNA construct will vary according tothe number of components (exogenous DNA, targeting sequences, selectablemarker gene) and the length of each. The entire construct length willgenerally be at least 20 nucleotides. In a construct in which theexogenous DNA has sufficient homology with genomic DNA to undergohomologous recombination, the construct will include a single component,the exogenous DNA. In this embodiment, the exogenous DNA, because of itshomology, serves also to target integration into genomic DNA andadditional targeting sequences are unnecessary. Such a construct isuseful to knock out, replace or repair a resident DNA sequence, such asan entire gene, a gene portion, a regulatory element or portion thereofor regions of DNA which, when removed, place regulatory and structuralsequences in functional proximity. It is also useful when the exogenousDNA is a selectable marker.

In another embodiment, the DNA construct includes exogenous DNA and oneor more separate targeting sequences, generally located at both ends ofthe exogenous DNA sequence. Targeting sequences are DNA sequencesnormally present in the primary or secondary cell genome in the genomeof the cells as obtained [e.g., an essential gene, a nonessential geneor noncoding DNA, or present in the genome through a previousmodification]. Such a construct is useful to integrate exogenous DNAencoding a therapeutic product, such as a hormone, a cytokine, anantigen, an antibody, an enzyme, a clotting factor, a transport protein,a receptor, a regulatory protein, a structural protein, an anti-senseRNA, a ribozyme or a protein or a nucleic acid which does not occur innature. In particular, exogenous DNA can encode one of the following:Factor VIII, Factor IX, erythropoietin, alpha-1 antitrypsin, calcitonin,glucocerebrosidase, growth hormone, low density lipoprotein (LDL)receptor, IL-2 receptor and its antagonists, insulin, globin,immunoglobulins, catalytic antibodies, the interleukins, insulin-likegrowth factors, superoxide dismutase, immune responder modifiers,parathyroid hormone, interferons, nerve growth factors, tissueplasminogen activators, and colony stimulating factors. Such a constructis also useful to integrate exogenous DNA which is a therapeuticproduct, such as DNA sequences sufficient for sequestration of a proteinor nucleic acid in the transfected primary or secondary cell, DNAsequences which bind to a cellular regulatory protein, DNA sequenceswhich alter the secondary or tertiary chromosomal structure and DNAsequences which are transcriptional regulatory elements into genomic DNAof primary or secondary cells.

In a third embodiment, the DNA construct includes exogenous DNA,targeting DNA sequences and DNA encoding at least one selectable marker.In this third embodiment, the order of construct components can be:targeting sequences-exogenous DNA-DNA encoding a selectablemarker(s)-targeting sequences. In this embodiment, one or moreselectable markers are included in the construct, which makes selectionbased on a selectable phenotype possible. Cells that stably integratethe construct will survive treatment with the selective agent; a subsetof the stably transfected cells will be HR cells, which can beidentified by a variety of techniques, including PCR, Southernhybridization and phenotypic screening.

In a fourth embodiment, the order of components in the DNA construct canbe: targeting sequence-selectable marker 1--targetingsequence--selectable marker 2. In this embodiment selectable marker 2displays the property negative selection. that is, the gene product ofselectable marker 2 can be selected against by growth in an appropriatemedia formulation containing an agent (typically a drug or metaboliteanalog) which kills cells expressing selectable marker 2. Recombinationbetween the targeting sequences flanking selectable marker 1 withhomologous sequences in the host cell genome results in the targetedintegration of selectable marker 1, while selectable marker 2 is notintegrated. Such recombination events generate cells which are stablytransfected with selectable marker 1 but not stably transfected withselectable marker 2, and such cells can be selected for by growth in themedia containing the selective agent which selects for selectable marker1 and the selective agent which selects against selectable marker 2.

In all embodiments of the DNA construct, exogenous DNA can encode one ormore products, can be one or more therapeutic products or one or more ofeach, thus making it possible to deliver multiple products.

Replacement of a Regultory Sequence of a Gene by HomologousRecombination

As taught herein, gene targeting can be used to replace a gene'sexisting regulatory region with a regulatory sequence isolated from adifferent gene or a novel regulatory sequence synthesized by geneticengineering methods. Such regulatory sequences may be comprised ofpromoters, enhancers, Scaffold-attachment regions, negative regulatoryelements, transcriptional initiation sites, reguatory protein bindingsites or combinations of said sequences. (Alternatively, sequences whichaffect the structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules).

Several embodiments are possible. First, the targeting event may be asimple insertion of the regulatory sequence, placing the gene under thecontrol of the new regulatory sequence (for example, inserting a newpromoter or enhancer or both upstream of a gene). Second, the targetingevent may be a simple deletion of a regulatory element, such as thedeletion of a tissue-specific negative regulatory element. Third, thetargeting event may replace an existing element; for example, atissue-specific enhancer can be replaced by an enhancer that has broaderor different cell-type specificity than the naturally-occurringelements. In this embodiment the naturally occurring sequences aredeleted and new sequences are added. In all cases, the identification ofthe targeting event may be facilitated by the use of one or moreselectable marker genes that are contiguous with the targeting DNA,allowing for the selection of cells in which the exogenous DNA hasintegrated into the host cell genome. The identification of thetargeting event may also be facilitated by the use of one or more markergenes exhibiting the property of negative selection, such that thenegatively selectable marker is linked to the exogenous DNA, butconfigured such that the negatively selectable marker flanks thetargeting sequence, and such that a correct homologous recombinationevent with sequences in the host cell genome does not result in thestable integration of the negatively selectable marker. Markers usefulfor this purpose include the Herpes Simplex Virus thymidine kinase (TK)gene or the bacterial xanthine-guanine phosphoribosyltransferase (gpt)gene.

Transfection of Primary or Secondary Cells and Production of Clonal orHeterogenous Cell Strains

The method of the present invention is carried out as follows:Vertebrate tissue is first obtained; this is carried out using knownprocedures, such as punch biopsy or other surgical methods of obtaininga tissue source of the primary cell type of interest. For example, punchbiopsy is used to obtain skin as a source of fibroblasts orkeratinocytes. A mixture of primary cells is obtained from the tissue,using known methods, such as enzymatic digestion or explanting. Ifenzymatic digestion is used, enzymes such as collagenase, hyaluronidase,dispase, pronase, trypsin, elastase and chymotrypsin can be used.

The resulting primary cell mixture can be transfected directly or it canbe cultured first, removed from the culture plate and resuspended beforetransfection is carried out. Primary cells or secondary cells arecombined with exogenous DNA to be stably integrated into their genomesand, optionally, DNA encoding a selectable marker, and treated in orderto accomplish transfection. The exogenous DNA and selectablemarker-encoding DNA are each on a separate construct (e.g., pXGH5 andpcDNEO, see FIGS. 1 and 2) or on a single construct (e.g., pXGH301, seeFIG. 3) and an appropriate quantity of DNA to ensure that at least onestably transfected cell containing and appropriately expressingexogenous DNA is produced. In general, 0.1 to 500 μg DNA is used.

Using the present methods to introduce only a selectable marker gene,between 170 (1 in 588 starting cells treated by electroporation) and2000 (1 in 49 starting cells treated by microinjection) stablytransfected cells are generated per 100,000 starting cells. Using thepresent methods to introduce a therapeutic gene as well as a selectablemarker gene, between 7 (1 in 14,705 starting cells treated byelectroporation) and 950 (1 in 105 starting cells treated bymicroinjection) stably transfected cells are generated per 100,000starting cells. Of these stable transfectants, from 43 to 90% expressthe gene of therapeutic interest. Since only a single appropriatelyexpressing cell is required, it is clearly possible to use substantiallyfewer starting cells. Conversely, using transfection techniques whichare substantially less efficient than the present methods, it would notbe possible to obtain even a single such cell unless large amount of theindividual's tissue is used as the source of starting cells.

In one embodiment of the present method of producing transfected primaryor secondary cells, transfection is effected by electroporation, asdescribed in Example 1. Electroporation is carried out at appropriatevoltage and capacitance (and corresponding time constant) to result inentry of the DNA construct(s) into the primary or secondary cells.Electroporation can be carried out over a wide range of voltages (e.g.,50 to 2000 volts) and corresponding capacitance. As described herein,electroporation is very efficient if carried out at an electroporationvoltage in the range of 250-300 volts and a capacitance of 960 μFarads(see Example 1). Total DNA of approximately 0.1 to 500 82 g is generallyused. Total DNA of 60 μg and voltage of 250-300 volts with capacitanceof 960 μFarads for a time constant 14-20 of msec. has been used andshown to be efficient.

In another embodiment of the present method, primary or secondary cellsare transfected using microinjection. Alternatively, known methods suchas calcium phosphate precipitation, modified calcium phosphateprecipitation and polybrene precipitation, liposome fusion andreceptor-mediated gene delivery can be used to transfect cells. A stablytransfected cell is isolated and cultured and subcultivated, underculturing conditions and for sufficient time, to propagate the stablytransfected secondary cells and produce a clonal cell strain oftransfected secondary cells. Alternatively, more than one transfectedcell is cultured and subcultured, resulting in production of aheterogenous cell strain.

Transfected primary or secondary cells undergo a sufficient number ofdoublings to produce either a clonal cell strain or a heterogenous cellstrain of sufficient size to provide the therapeutic product to anindividual in effective amounts. In general, for example, 0.1 cm² ofskin is biopsied and assumed to contain 100,000 cells; one cell is usedto produce a clonal cell strain and undergoes approximately 27 doublingsto produce 100 million transfected secondary cells. If a heterogenouscell strain is to be produced from an original trnasfected population ofapproximately 100,000 cells, only 10 doublings are needed to produce 100million transfected cells.

The number of required cells in a transfected clonal or heterogenouscell strain is variable and depends on a variety of factors, includingbut not limited to, the use of the transfected cells, the functionallevel of the exogenous DNA in the transfected cells, the site ofimplantation of the transfected cells (for example, the number of cellsthat can be used is limited by the anatomical site of implantation), andthe age, surface area, and clinical condition of the patient. To putthese factors in perspective, to deliver therapeutic levels of humangrowth hormone in an otherwise healthy 10 kg patient with isolatedgrowth hormone deficiency, approximately one to five hundred milliontransfected fibroblasts would be necessary (the volume of these cells isabout that of the very tip of the patient's thumb).

Implantation of Clonal Cell Strains or Heteroyenous Cell Strains ofTransfected Secondary Cells

The homologously recombinant cells produced as described above areintroduced into an individual to whom the therapeutic product is to bedelivered, using known methods. The clonal cell strain or heterogenouscell strain is introduced into an individual, using known methods, usingvarious routes of administration and at various sites (e.g., renalsubcapsular, subcutaneous, central nervous system (includingintrathecal), intravascular, intrahepatic, intrasplanchnic,intraperitoneal (including intraomental), or intramuscularimplantation). Once implanted in the individual, the transfected cellsproduce the therapeutic product encoded by the exogenous DNA or areaffected by the exogenous DNA itself. For example, an individual who hasbeen diagnosed with Hemophilia B, a bleeding disorder that is caused bya deficiency in Factor IX, a protein normally found in the blood, is acandidate for a gene therapy cure. The patient has a small skin biopsyperformed; this is a simple procedure which can be performed on anout-patient basis. The piece of skin, approximately the size of amatchhead, is taken, for example, from under the arm and requires aboutone minute to remove. The sample is processed, resulting in isolation ofthe patient's cells (in this case, fibroblasts) and geneticallyengineered to produce the missing Factor IX. Based on the age, weight,and clinical condition of the patient, the required number of cells aregrown in large-scale culture. The entire process usually requires 4-6weeks and, at the end of that time, the appropriate number ofgenetically-engineered cells are introduced into the individual, onceagain as an out-patient (e.g., by injecting them back under thepatient's skin). The patient is now capable of producing his or her ownFactor IX and is no longer a hemophiliac.

As this example suggests, the cells used will generally bepatient-specific genetically-engineered cells. It is possible, however,to obtain cells from another individual of the same species or from adifferent species. Use of such cells might require administration of animmunosuppressant, alteration of histocompatibility antigens, or use ofa barrier device to prevent rejection of the implanted cells. For manydiseases, this will be a one-time treatment and, for others, multiplegene therapy treatments will be required.

Uses of Homologously Recombinant Primary and Secondary Cells and CellStrains

HR primary or secondary cells or cell strains have wide applicability asa vehicle or delivery system for therapeutic products, such as enzymes,hormones, cytokines, antigens, antibodies, clotting factors, anti-senseRNA, regulatory proteins, transcription proteins, receptors, structuralproteins, ribozymes, novel (non-naturally occurring) proteins andnucleic acid products, and engineered DNA. For example, transfectedprimary or secondary cells can be used to supply a therapeutic protein,including, but not limited to, Factor VIII, Factor IX, erythropoietin,alpha-1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, lowdensity lipoprotein (LDL) receptor, IL-2 receptor and its antagonists,insulin, globin, immunoglobulins, catalytic antibodies, theinterleukins, insulin-like growth factors, superoxide dismutase, immuneresponder modifiers, parathyroid hormone, interferons, nerve growthfactors, tissue plasminogen activators, and colony stimulating factors.Alternatively, transfected primary and secondary cells can be used toimmunize an individual (i.e., as a vaccine). In the case where targetingis utilized to introduce additional DNA sequences into the genome, theability to pre-select the integration site offers many advantages ascompared to random integration. For example, the additional sequencescan be directed to a region of the genome that allows appropriateexpression and to regions that are distant from oncogenes. The sequencescan be targeted to non-essential or essential genes or to non-codingsequences as desired. The choice of site can be determined based on theabove considerations or based on known integration sites ofwell-characterized, appropriately-functioning transfected cells.

The wide variety of uses of cell strains of the present invention canperhaps most conveniently be summarized as shown below. The cell strainscan be used to deliver the following therapeutic products.

1. a secreted protein with predominantly systemic effects;

2. a secreted protein with predominantly local effects;

3. a membrane protein imparting new or enhanced cellular responsiveness;

4. membrane protein facilitating removal of a toxic product;

5. a membrane protein marking or targeting a cell;

6. an intracellular protein;

7. an intracellular protein directly affecting gene expression;

8. an intracellular protein with autolytic effects;

9. gene product-engineered DNA which binds to or sequesters a regulatoryprotein;

10. a ribozyme; and

11. antisense-engineered RNA to inhibit gene expression.

The transfected primary or secondary cells of the present invention canbe used to administer therapeutic proteins (e.g., hormones, enzymes,clotting factors) which are presently administered intravenously,intramuscularly or subcutaneously, which requires patient cooperationand, often, medical staff participation. When transfected primary orsecondary cells are used, there is no need for extensive purification ofthe polypeptide before it is administered to an individual, as isgenerally necessary with an isolated polypeptide. In addition,transfected primary or secondary cells of the present invention producethe therapeutic product as it would normally be produced.

An advantage to the use of transfected primary or secondary cells of thepresent invention is that by controlling the number of cells introducedinto an individual, one can control the amount of the product deliveredto the body. In addition, in some cases, it is possible to remove thetransfected cells if there is no longer a need for the product. Afurther advantage of treatment by use of transfected primary orsecondary cells of the present invention is that production of thetherapeutic product can be regulated, such as through the administrationof zinc, steroids or an agent which affects translation or transcriptionof a protein product or nucleic acid product or affects the stability ofa nucleic acid product.

The subject invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES EXAMPLE 1

Stable Transfection of Primary or Secondary Human Cells

1. Generation of a Construct (XGH 301) Containing Both the Human GrowthHormone and Neomycin Resistance Genes

pXGH301 was constructed by a two-step procedure. The SaII-ClaI fragmentfrom pBR322 (positions 23-651 in pBR322) was isolated and inserted intoSaII-ClaI digested pcDNEO, introducing a BamHI site upstream of the SV40early promoter region of pcDNEO. This plasmid, pBNEO was digested withBamHI, and the 2.1 kb fragment containing the neo gene under the controlof the SV40 early promoter, was isolated and inserted into BamHIdigested pXGH5. A plasmid with a single insertion of the 2.1 kb BamHIfragment was isolated in which neo and hGH are transcribed in the samedirection relative to each other. This plasmid was designated pXGH301(FIG. 3).

2. Stable Transfection of Primary or Secondary Human Cells

Exponentially growing or early stationary phase fibroblasts aretrypsinized and rinsed from the plastic surface with nutrient medium. Analiquot of the cell suspension is removed for counting, and theremaining cells are subjected to centrifugation. The supernatant isaspirated and the pellet is resuspended in 5 ml of electroporationbuffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mMdextrose). The cells are recentrifuged, the supernatant aspirated, andthe cells resuspended in electroporation buffer containing 1 mg/mlacetylated bovine serum albumin. The final cell suspension containsapproximately 3×10⁶ cells/ml. Electroporation should be performedimmediately following resuspension.

Supercoiled plasmid DNA is added to a sterile cuvette with a 0.4 cmelectrode gap (Bio-Rad). The final DNA concentration is generally atleast 120 μg/ml. 0.5 ml of the cell suspension (containing approximately1.5×10⁶ cells) is then added to the cuvette, and the cell suspension andDNA solutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 msec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (as above with 15% calf serum) in a 10cm dish and incubated as described above. The following day, the mediais aspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hrs. Subculture of cells to determine cloning efficiencyand to select for G418-resistant colonies is performed the followingday. Cells are trypsinized, counted and plated; typically, fibroblastsare plated at 10³ cells/10 cm dish for the determination of cloningefficiency and at 1-2×10⁴ cells/10 cm dish for G418 selection.

Human fibroblasts are selected for G418 resistance in medium consistingof 300-400 μg/ml G418 (Geneticin, disulfate salt with a potency ofapproximately 50%; Gibco) in fibroblasts nutrient media (with 15% calfserum). Cloning efficiency is determined in the absence of G418. Theplated cells are incubated for 12-14 days, at which time colonies arefixed with formalin, stained with crystal violet and counted (forcloning efficiency plates) or isolated using cloning cylinders (for G418plates). Electroporation and selection of rabbit fibroblasts isperformed essentially as described for human fibroblasts, with theexception of the selection conditions used. Rabbit fibroblasts areselected for G418 resistance in medium containing 1 gm/ml G418.

Fibroblasts were isolated from freshly excised human foreskins. Cultureswere seeded at 50,000 cells/cm² in DMEM+10% calf serum. When culturesbecame confluent fibroblasts were harvested by trypsinization andtransfected by electroporation. Electroporation conditions wereevaluated by transfection with the plasmid pcDNEO. A representativeelectroporation experiment using near optimal conditions (60 μg ofplasmid pcDNEO at an electroporation voltage of 250 volts and acapacitance setting of 960 μFarads) resulted in one G418^(r) coloney per588 treated cells (0.17% of all cells treated), or one G418^(r) colonyper 71 clonable cells (1.4%).

When nine separate electroporation experiments at near optimalconditions (60 μg of plasmid pcDneo at an electroporation voltage of 300volts and a capacitance setting of 960 μFarads) were performed, anaverage of one G418^(r) colony per 1,899 treated cells (0.05%) wasobserved, with a range of 1/882 to 1/7,500 treated cells. Thiscorresponds to an average of one G418^(r) colony per 38 clonable cells(2.6%).

Low passage primary human fibroblasts were converted to hGH expressingcells by co-transfection with plasmids pcDNEO and pXGH5. Typically, 60μg of an equimolar mixture of the two plasmids were transfected at nearoptimal conditions (electroporation voltage of 300 volts and acapacitance setting of 960 μFarads). The results of such an experimentresulted in one G418^(r) colony per 14,705 treated cells.

hGH expression data for these and other cells isolated under identicaltransfection conditions are summarized below. Ultimately, 98% of allG418^(r) colonies could be expanded to generate mass cultures.

    ______________________________________                                        Number of G418.sup.r Clones                                                                     154                                                         Analyzed                                                                      Number of G418.sup.r /hGH                                                                               65                                                  Expressing Clones                                                             Average hGH Expression                                                                                      2.3 μg hGH/10.sup.6 Cells/24 hr              Level                                                                         Maximum hCH Expression                                                                                      23.0 μg hGH/10.sup.6 Cells/24 hr             Level                                                                         ______________________________________                                    

EXAMPLE 2

Generation of a Construct Useful for Selection of Gene Targeting Eventsin Human Cells

One approach to selecting the targeted events is by genetic selectionfor the loss of a gene function due to the integration of transfectingDNA. The human HPRT locus encodes the enzyme hypoxanthine-phosphoribosyltransferase. hprt⁻ cells can be selected for by growth in mediumcontaining the nucleoside analog 6-thioguanine (6-TG): cells with thewild-type (HPRT+) allele are killed by 6-TG, while cells with mutant(hprt⁻) alleles can survive. Cells harboring targeted events whichdisrupt HPRT gene function are therefore selectable in 6-TG medium.

To construct a plasmid for targeting to the HPRT locus, the 6.9 kbHindIII fragment extending from positions 11,960-18,869 in the HPRTsequence (Genebank name HUMHPRTB; Edwards, A. et al., Genomics 6:593-608(1990)) and including exons 2 and 3 of the HPRT gene, is subcloned intothe HindIII site of pUC12. The resulting clone is cleaved at the uniqueXhoI site in exon 3 of the HPRT gene fragment and the 1.1 kb SalI-XhoIfragment containing the neo gene from pMClNeo (Stratagene) is inserted,disrupting the coding sequence of exon 3. One orientation, with thedirection of neo transcription opposite that of HPRT transcription waschosen and designated pE3Neo. The replacement of the normal HPRT exon 3with the neo-disrupted version will result in an hprt⁻, 6-TG resistantphenotype. Such cells will also be G418 resistant.

EXAMPLE 3

Gene Targeting in an Established Human Fibroblast Cell Line

Gene targeting has previously only been reported for immortalized tissueculture cell lines. As a positive control for targeting and to establishthat pE3Neo functions properly in gene targeting, the human fibrosarcomacell line HT1080 (ATCC CCL 121) was transfected with pE3Neo byelectroporation.

HT1080 cells were maintained in HAT (hypoxanthine/aminopterin/xanthine)supplemented DMEM with 15% calf serum (Hyclone) prior toelectroporation. Two days before electroporation, the cells are switchedto the same medium without aminopterin. Exponentially growing cells weretrypsinized and diluted in DMEM/15% calf serum, centrifuged, andresuspended in PBS (phosphate buffered saline) at a final cell bolume of13.3 million cells per ml. pE3Neo is digested with HindIII, separatingthe 8 kb HPRT-neo fragment from the pUC12 backbone, purified by phenolextraction and ethanol precipitation, and resuspended at a concentrationof 600 μg/ml. 50 μl (30 μg) was added to the electroporation cuvette(0.4 cm electrode gap; Bio-Rad Laboratories), along with 750 μl of thecell suspension (10 million cells). Electroporation was at 450 volts,250 μFarads (Bio-Rad Gene Pulser; Bio-Rad Laboratories). The contents ofthe cuvette were immediately added to DMEM with 15% calf serum to yielda cell suspension of 1 million cells per 25 ml media. 25 ml of thetreated cell suspension was plated onto 150 mm diameter tissue culturedishes and incubated at 37° C., 5% CO₂. 24 hrs later, a G418 solutionwas added directly to the plates to yield a final concentration of 800μg/ml G418. Five days later the media was replaced with DMEM/15% calfserum/800 μg/ml G418. Nine days after electroporation, the media wasrepalced with DMEM/15% calf serum/800 μg/ml G418 and 10 μM6-thioguanine. Colonies resistant to G418 and 6-TG were picked usingcloning cylinders 14-16 days after the dual selection was initiated.

The results of five representative targeting experiments in HT1080 cellsare shown in the table below.

    ______________________________________                                                                Number of                                                                               G4l8.sup.r                                  Transfection   Treated Cells                                                                                6-TG.sup.r Clones                               ______________________________________                                        1             1 × 10.sup.7                                                                      32                                                    2                          28times. 10.sup.7                                  3                          24times. 10.sup.7                                  4                          32times. 10.sup.7                                  5                          66times. 10.sup.7                                  ______________________________________                                    

For transfection 5, control plates designed to determine the overallyield of G418^(r) colonies indicated that 33,700 G418^(r) colonies couldbe generated from the initial 1×10⁷ treated cells. Thus, the ratio oftargeted to non-targeted events is 66/33,700, or 1 to 510. In the fiveexperiments combined, targeted events arise at a frequency of 3.6×10⁻⁶,or 0.00036% of treated cells.

Restriction enzyme and Southern hybridization experiments using probesderived from the neo and HPRT genes localized the neo gene to the HPRTlocus at the predicted site within HPRT exon 3.

EXAMPLE 4

Gene Targeting in Primary and Secondary Human Skin Fibroblasts

pE3Neo is digested with HindIII, separating the 8 kb HPRT-neo fragmentfrom the pUC12 backbone, and purified by phenol extraction and ethanolprecipitation. DNA was resuspended at 2 mg/ml. Three million secondaryhuman foreskin fibroblasts cells in a volume of 0.5 ml wereelectroporated at 250 volts and 960 μFarads, with 100 μg of HindIIIpE3Neo (50 μl ). Three separate transfections were performed, for atotal of 9 million treated cells. Cells are processed and selected forG418 resistance as described in Example 1, except that 500,000 cells per150 mm culture dish are plated for G418 selection. After 10 days underselection, the culture medium is replaced with human fibroblast nutrientmedium containing 400 μg/ml G418 and 10 μM 6-TG. Selection with the twodrug combination is continued for 10 additional days. Plates are scannedmicroscopically to localize human fibroblast colonies resistant to bothdrugs. The fraction of G418^(r) t-TG^(r) colonies is 4 per 9 milliontreated cells. These colonies constitute 0.0001% (or 1 in a million) ofall cells capable of forming colonies. Control plates designed todetermine the overall yield of G418^(r) colonies indicated that 2,850G418 colonies could be generated from the initial 9×10⁶ treated cells.Thus, the ratio of targeted to non-targeted events is 4/2,850, or 1 to712. Restriction enzyme and Southern hybridization experiments usingprobes derived from the neo and HPRT genes were used to localize the neogene to the HPRT locus at the predicted site within HPRT exon 3 anddemonstrate that targeting had occurred in these four clonal cellstrains. Colonies resistant to both drugs have also been isolated bytransfecting primary cells (1/3.0×10⁷).

EXAMPLE 5

Generation of a Construct for Targeted Insertion of a Gene ofTherapeutic Interest into the Human Genome and its Use in Gene Targeting

A variant of pE3Neo, in which a gene of therapeutic interest is insertedwithin the HPRT coding region, adjacent to or near the neo gene, can beused to target a gene of therapeutic interest to a specific position ina recipient primary or secondary cell genome. Such a variant of pE3Neocan be constructed for targeting the hGH gene to the HPRT locus.

pXGH5 is digested with EcoRI and the 4.1 kb fragment containing the hGHgene and linked mouse metallothionein (mMT) promoter is isolated. TheEcoRI overhangs are filled in with the Klenow fragment from E. coli DNApolymerase. Separately, pE3Neo is digested with XhoI, which cuts at thejunction of the neo fragment and HPRT exon 3 (the 3' junction of theinsertion into exon 3). The XhoI overhanging ends of the linearizedplasmid are filled in with the Klenow fragment from E. coli DNApolymerase, and the resulting fragment is ligated to the 4.1 kbblunt-ended hGH-mMT fragment. Bacterial colonies derived from theligation mixture are screened by restriction enzyme analysis for asingle copy insertion of the hGH-mMT fragment and one orientation, thehGH gene transcribed in the same direction as the neo gene, is chosenand designated pE3Neo/hGH. pE3Neo/hGH is digested with HindIII,releasing the 12.1 kb fragment containing HPRT, neo and mMT-hGHsequences. Digested DNA is treated and transfected into primary orsecondary human fibroblasts as described in Example 4. G418^(r) TG^(r)colonies are selected and analyzed for targeted insertion of the mMT-hGHand neo sequences into the HPRT gene as described in Example 4.Individual colonies are assayed for hGH expression using a commerciallyavailable immunoassay (Nichols Institute).

EXAMPLE 6

Modification of DNA Termini to Enhance Targeting

Several lines of evidence suggest that 3'-overhanging ends are involvedin certain homologous recombination pathways of E. coli, bacteriophage,S. cerevisiae, and Xenopus laevis. In Xenopus laevis oocytes, moleculeswith 3'-overhanging ends of several hundred base pairs in lengthunderwent recombination with similarly treated molecules much morerapidly after microinjection than molecules with very short overhangs (4bp) generated by restriction enzyme digestion. In yeast, the generationof 3'-overhanging ends several hundred base pairs in length appears tobe a rate limiting step in meiotic recombination. No evidence for aninvolvement of 3'-overhanging ends in recombination in human cells hasbeen reported, and in no case have modified DNA substrates of any sortbeen shown to promote targeting (one form of homologous recombination)in any species. In human cells, the effect of 3'-overhanging ends ontargeting is untested. The experiment described in the following examplesuggests that 5'-overhanging ends are most effective for targeting inprimary and secondary human fibroblasts.

There have been no reports on the enhancement of targeting by modifyingthe ends of the transfecting DNA molecules. This example serves toillustrate that modification of the ends of linear DNA molecules, byconversion of the molecules' termini from a double-stranded form to asingle-stranded form, can stimulate targeting into the genome of primaryand secondary human fibroblasts.

1100 μg of plasmid pE3Neo (Example 2) is digested with HindIII. This DNAcan be used directly after phenol extraction and ethanol precipitation,or the 8 kb HindIII fragment containing only HPRT and the neo gene canbe separated away from the pUC12 vector sequences by gelelectrophoresis. ExoIII digestion of the HindIII digested DNA results inextensive exonucleolytic digestion at each end, initiating at each free3' end, and leaving 5'-overhanging ends. The extent of exonucleolyticaction and, hence, the length of the resulting 5'-overhangings, can becontrolled by varying the time of ExoIII digestion. ExoIII digestion of100 μg of HindIII digested pE3Neo is carried out according to thesupplier's recommended conditions, for times of 30 sec, 1 min, 1.5 min,2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, and 5 min. To monitorthe extent of digestion an aliquot from each time point, containing 1 μgof ExoIII treated DNA, is treated with mung bean nuclease (Promega),under conditions recommended by the supplier, and the samplesfractionated by gel electrophoresis. The difference in size betweennon-treated, HindIII digested pE3Neo and the same molecules treated withExoIII and mung bean nuclease is measured. This size difference dividedby two gives the average length of the 5'-overhang at each end of themolecule. Using the time points described above and digestion at 30°,the 5'-overhangs produced should range from 100 to 1,000 bases. 60 μg ofExoIII treated DNA (total HindIII digest of pE3NEO) from each time pointis purified and electroporated into primary or secondary humanfibroblasts under the conditions described in Example 4. The degree towhich targeting is enhanced by each ExoIII treated preparation isquantified by counting the number of G418^(r) 6-TG^(r) colonies andcomparing these numbers to targeting with HindIII digested pE3Neo thatwas not treated with ExoIII.

The effect of 3'-overhanging ends can also be quantified using ananalogous system. In this case HindIII digested pE3Neo is treated withbacteriophase T7 gene 6 exonuclease (United States Biochemicals) forvarying time intervals under the supplier's recommended conditions.Determination of the extent of digestion (average length of 3'-overhangproduced per end) and electroporation conditions are as described forExoIII teated DNA. The degree to which targeting is enhanced by each T7gene 6 exonuclease treated preparation is quantified by counting thenumber of G418^(r) 6-TG^(r) colonies and comparing these numbers totargeting with HindII digested pE3NEO that was not treated with T7 gene6 exonuclease.

Other methods for generating 5' and 3' overhanging ends are possible,for example, denaturation and annealing of two linear molecules thatpartially overlap with each other will generate a mixture of molecules,each molecule having 3'-overhangs at both ends or 5'-overhangs at bothends, as well as reannealed fragments indistinguishable from thestarting linear molecules. The length of the overhangs is determined bythe length of DNA that is not in common between the two DNA fragments.

Example 7

Construction of Targeting Plasmids For Placing the Human ErythropoietinGene Under the Control of the Mouse Metallothionein Promoter in Primaryand Secondary Human Fibroblasts

The following serves to illustrate one embodiment of the presentinvention, in which thenormal positive and negative regulatory sequencesupstream of the human erythropoietin (EPO) gene are altered to allowexpression of human erythropoietin in primary or secondary humanfibroblast strains, which do not express EPO in significant quantitiesas obtained.

A region lying exclusively upstream of the human EPO coding region canbe amplified by PCR. Three sets of primers useful for this purpose weredesigned after analysis of the published human EPO [Genbank designationHUMERPA; Lin, F-K., et al., Proc. Natl. Acad. Sci or USA. 82:7580-7584(1985)]. These primer pairs can amplify fragments of 609, 603, or 590bp.

          HUMERPA                      Fragment                                   Primer                                                                              Coordinate Sequence          Size                                       ______________________________________                                        F1    2->20      5' AGCTTCTGGGCTTCCAGAC                                                        (SEQ. ID. NO. 1)                                             R2    610->595   5' GGGGTCCCTCAGCGAC                                                                             609 bp                                                      (SEQ. ID. NO. 2)                                             F2    8->24      5' TGGGCTTCCAGACCCAG                                                          (SEQ. ID. NO. 3)                                             R2    610->595   5' GGGGTCCCTCAGCGAC                                                                             603 bp                                                      (SEQ. ID. NO. 2)                                             F3    21->40     5' CCAGCTACTTTGCGGAACTC                                                       (SEQ. ID. NO. 4)                                             R3    610->595   5' GGGGTCCCTCAGCGAC                                                                             590 bp                                                      (SEQ. ID. NO. 2)                                             ______________________________________                                    

The three fragments overlap substantially and are interchangeable forthe present purposes. The 609 bp fragment, extending from -623 to -14relative to the translation start site (HUMERPA nucleotide positions 2to 610), is ligated at both ends with ClaI linkers. The resultingClaI-linked fragment is digested with ClaI and inserted into the ClaIsite of pBluescriptIISK/+ (Stratagene), with the orientation such thatHUMERPA nucleotide position 610 is adjacent to the SaII site in theplasmid polylinker). This plasmid, p5'EPO, can be cleaved, separately,at the unique FspI or SfiI sites in the EPO upstream fragment (HUMERPAnucleotide positions 150 and 405, respectively) and ligated to the mousemetallotheionein promoter. Typically, the 1.8 kb EcoRI-BgIII from themMT-I gene [containing no mMT coding sequences; Hamer, D. H. and WallingM., J. Mol. Appl. Gen. 1:273-288 (1982); this fragment can also beisolated by known methods from mouse genomic DNA using PCR primersdesigned from analysis of mMT sequences available from Genbank; i.e.,MUSMTI, MUSMTIP, MUSMTIPRM] is made blunt-ended by known methods andligated with SfiI digested (also made blunt-ended) or FspI digestedp5'EPO. The orientations of resulting clones are analyzed and those inwhich the former mMT BgIII site is proximal to the SaII site in theplasmid polylinker are used for targeting primary and secondary humanfibroblasts. This orientation directs mMT transcription towards HUMERPAnucleotide position 610 in the final construct. The resulting plasmidsare designated p5'EPO-mMTF and p5'EPO-mMTS for the mMT insertions in theFspI and SfiI sites, respectively.

Additional upstream sequences are useful in cases where it will bedesirable to modify, delete and/or replace negative regulatory elementsor enhancers that lie upstream of the initial target sequence. In thecase of EPO, a negative regulatory element that inhibits EPO expressionin extrahepatic and extrarenal tissues [Semenza, G.L. et al., Mol. Cell.Biol. 10:930-938 (1990)] can be deleted. A series of deletions withinthe 6 kb fragment are prepared. The deleted regions may be replaced withan enhancer with broad host-cell activity [e.g. an enhancer from theCytomegalovirus (CMV)].

The orientation of the 609 bp 5'EPO fragment in the pBluescriptIISK/+vector was chosen since the HUMERPA sequences are preceded on their 5'end by a BamHI (distal) and HindIII site (proximal). Thus, a 6 kbBamHI-HindIII fragment normally lying upstream of the 609 bp fragment[Semenza, G. L. et al., Mol. Cell. Biol. 10:930-938 (1990)] can beisolated from genomic DNA by known methods. For example, abacteriophage, cosmid, or yeast artificial chromosome library could bescreened with the 609 bp PCR amplified fragment as a probe. The desiredclone will have a 6 kb BamHI-HindIII fragment and its identity can beconfirmed by comparing its restriction map from a restriction map aroundthe human EPO gene determined by known methods. Alternatively,constructing a restriction map of the human genome upstream of the EPOgene using the 609 bp fragment as a probe can identify enzymes whichgenerate a fragment originating between HUMERPA coordinates 2 and 609and extending past the upstream BamHI site; this fragment can beisolated by gel electrophoresis from the appropriate digest of humangenomic DNA and ligated into a bacterial or yeast cloning vector. Thecorrect clone will hybridize to the 609 bp 5'EPO probe and contain a 6kb BamHI-HindIII fragment. The isolated 6 kb fragment is inserted in theproper orientation into p5'EPO, p5'EPO-mMTF, or p5'EPO-mMTS (such thatthe HindIII site is adjacent to HUMERPA nucleotide position 2).Additional upstream sequences can be isolated by known methods, usingchromosome walking techniques or by isolation of yeast artificialchromosomes hybridizing to the 609 bp 5'EPO probe.

The cloning strategies described above allow sequences upstream of EPOto be modified in vitro for subsequent targeted transfection of primaryand secondary human fibroblasts. The strategies describe simpleinsertions of the mMT promoter, as well as deletion of the negativeregulatory region, and deletion of the negative regulatory region andreplacement with an enhancer with broad host-cell activity.

EXAMPLE 8

Targeting to Sequences Flanking, the Human EPO Gene and Isolation ofTargeted Primary and Secondary Human Fibroblasts by Screening

For targeting, the plasmids are cut with restriction enzymes that freethe insert away from the plasmid backbone. In the case of p5'EPO-mMTS,HindIII and SaII digestion releases a targeting fragment of 2.4 kb,comprised of the 1.8 kb mMT promoter flanked on the 5' and 3' sides by405 bp and 204 base pairs, respectively, of DNA for targeting thisconstruct to the regulatory region of the EPO gene. This DNA or the 2.4kb targeting fragment alone is purified by phenol extraction and ethanolprecipitation and transfected into primary or secondary humanfibroblasts under the conditions described in Example 4. Transfectedcells are plated onto 150 mm dishes in human fibroblast nutrient medium.48 hours later the cells are plated into 24 well dishes at a density of10,000 cells/cm2 [approximately 20,000 cells per well; if targetingoccurs at a rate of 1 event per 10⁶ clonable cells (Example 4, thenabout 50 wells would need to be assayed to isolate a single expressingcolony]. Cells in which the transfecting DNA has targeted to thehomologous region upstream of EPO will express EPO under the control ofthe mMT promoter. After 10 days, whole well supernatants are assayed forEPO expression using a commercially available immunoassay kit (Amgen).Clones from wells displaying EPO synthesis are isolated using knownmethods, typically by assaying fractions of the heterogenous populationsof cells separated into individual wells or plates, assaying fractionsof these positive wells, and repeating as needed, ultimately isolatingthe targeted colony by screening 96-well microtiter plates seeded at onecell per well. DNA from entire plate lysates can also be analyzed by PCRfor amplification of a fragment using a mMT specific primer inconjunction with a primer lying upstream of HUMERPA nucleotideposition 1. This primer pair should amplify a DNA fragment of a sizeprecisely predicted based on the DNA sequence. Positive plates aretrypsinized and replated at successively lower dilutions, and the DNApreparation and PCR steps repeated as needed to isolate targeted cells.

EXAMPLE 9

Targeting to Sequences Flanking the Human EPO Gene and Isolation ofTargeted Primary and Secondary Human Fibroblasts by a Positive or aCombined Positive/Negative Selection System

The strategy for constructing p5'EPO-mMTF, p5'EPO-mMTS, and derivativesof such with the additional upstream 6 kb BamHI-HindIII fragment can befollowed with the additional step of inserting the neo gene adjacent tothe mMT promoter. In addition, a negative selection marker, for example,gpt [from pMSG (Pharmacia) or another suitable source], can be insertedadjacent to the HUMERPA sequences in the pBluescriptIISK/+ polylinker.In the former case, G418^(r) colonies are isolated and screened by PCRamplification or restriction enzyme and Southern hybridization analysisof DNA prepared from pools of colonies to identify targeted colonies. Inthe latter case, G418^(r) colonies are placed in medium containing6-thioxanthine to select against the integration of the gpt gene[Besnard, C. et al., Mol. Cell. Biol. 7:4139-4141 (1987)]. In addition,the HSV-TK gene can be placed on the opposite side of the insert as gpt,allowing selection for neo and against both gpt and TK by growing cellsin human fibroblast nutrient medium containing 400 μg/ml G418, 100 μM6-thioxanthine, and 25 μg/ml gancyclovir. The double negative selectionshould provide a nearly absolute selection for true targeted events andSouthern blot analysis provides an ultimate confirmation.

EXAMPLE 10

Construction of Targeting Plasmids For Placing the Human Growth HormoneGene Under the Control of the Mouse Metallothionein Promoter in PrimaryHuman Fibroblasts

The following example serves to illustrate one embodiment of the presentinvention, in which the normal regulatory sequences upstream of thehuman growth hormone gene are altered to allow expression of humangrowth hormone in primary or secondary human fibroblast strains.

Targeting molecules similar to those described in Example 7 fortargeting to the EPO gene regulatory region are generated using clonedDNA fragments derived from the 5' end of the human growth hormone Ngene. An approximately 1.8 kb fragment spanning HUMGHCSA (Genbank Entry)nucleotide positions 3787-5432 (the positions of two EcoNI sites whichgenerate a convenient sized fragment for cloning or for diagnosticdigestion of subclones involving this fragment) is amplified by PCRprimers designed by analysis of the HUMGHCSA sequence in this region.This region extends from the middle of hGH gene N intron 1 to anupstream position approximately 1.4 kb 5' to the translational startsite. pUC12 is digested with EcoRI and BamHI, treated with Klenow togenerate blunt ends, and recircularized under dilute conditions,resulting in plasmids which have lost the EcoRI and BamHI sites. Thisplasmid is designated pUC12XEB. HindIII linkers are ligated onto theamplified hGH fragment and the resulting fragment is digested withHindIII and ligated to HindIII digested pUC12XEB. The resulting plasmid,pUC12XEB-5'hGH, is digested with EcoRI and BamHI, to remove a 0.5 kbfragment lying immediately upstream of the hGH transcriptionalinitiation site. The digested DNA is ligated to the 1.8 kb EcoRI-BglIIfrom the mMT-I gene [containing no mMT coding sequences; Hamer, D. H.and Walling, M., J. Mol. Appl. Gen.1:273-288 (1982); the fragment canalso be isolated by known methods from mouse genomic DNA using PCRprimers designed from analysis of mMT sequences available from Genbank;i.e., MUSMTI, MUSMTIP, MUSMTIPRM]. This plasmid p5'hGH-mMT has the mMTpromoter flanked on both sides by upstream hGH sequences.

The cloning strategies described above allow sequences upstream of hGHto be modified in vitro for subsequent targeted transfection of primaryand secondary human fibroblasts. The strategy described a simpleinsertion of the mMT promoter. Other strategies can be envisioned, forexample, in which an enhancer with broad host-cell specificity isinserted upstream of the inserted mMT sequence.

EXAMPLE 11

Targeting to Sequences Flanking the Human hGH Gene and Isolation ofTargeted Primary and Secondary Human Fibroblasts by Screening

For targeting, the plasmids are cut with restriction enzymes that freethe insert away from the plasmid backbone. In the case of p5'hGH-mMT,HindIII digestion releases a targeting fragment of 2.9 kb, comprised ofthe 1.8 kb mMT promoter flanked on the 5' end 3' sides by DNA fortargeting this construct to the regulatory region of the hGH gene. ThisDNA or the 2.9 kb targeting fragment alone is purified by phenolextraction and ethanol precipitation and transfected into primary orsecondary human fibroblasts under the conditions described in Example 1.Transfected cells are plated onto 150 mm dishes in human fibroblastnutrient medium. 48 hours later the cells are plated into 24 well dishesat a density of 10,000 cells/cm² [approximately 20,000 cells per well;if targeting occurs at a rate of 1 event per 10⁶ clonable cells (Example4), then about 50 wells would need to be assayed to isolate a singleexpressing colony]. Cells in which the transfecting DNA has targeted tothe homologous region upstream of hGH will express hGH under the controlof the mMT promoter. After 10 days, whole well supernatants are assayedfor hGH expression using a commercially available immunoassay kit(Nichols). Clones from wells displaying hGH synthesis are isolated usingknown methods, typically by assaying fractions of the heterogenouspopulations of cells separated into individual wells or plates, assayingfractions of these positive wells, and repeating as needed, ultimatelyisolated the targeted colony by screening 96-well microtiter platesseeded at one cell per well. DNA from entire plate lysates can also beanalyzed by PCR for amplification of a fragment using a mMT specificprimer in conjunction with a primer lying downstream of HUMGHCSAnucleotide position 5,432. This primer pair should amplify a DNAfragment of a size precisely predicted based on the DNA sequence.Positive plates are trypsinized and replated at successively lowerdilutions, and the DNA preparation and PCR steps repeated as needed toisolate targeted cells.

EXAMPLE 12

Targeting to Sequences Flanking, the Human GH Gene and Isolation ofTargeted Primary and Secondary Human Fibroblasts by a Positive or aCombined Positive/Negative Selection System

The strategy for constructing p5'hGH-mMT can be followed with theadditional step of inserting the neo gene adjacent to the mMT promoter.In addition, a negative selection marker, for example, gpt [from pMSG(Pharmacia) or another suitable source], can be inserted adjacent to theHUMGHCSA sequences in the pUC12 polylinker. In the former case, G418^(r)colonies are isolated and screened by PCR amplification or restrictionenzyme and Southern hybridization analysis of DNA prepared from pools ofcolonies to identify targeted colonies. In the latter case, G418^(r)colonies are placed in medium containing thioxanthine to select againstthe integration of the gpt gene [Besnard, C. et al., Mol. Cell. Biol.7:4139-4141 (1987)]. In addition, the HSV-TK gene can be placed on theopposite side of the insert as gpt, allowing selection for neo andagainst both gpt and TK by growing cells in human fibroblast nutrientmedium containing 400 μg/ml G418, 100 μM 6-thioxanthine, and 25 μg/mlgancyclovir. The double negative selection should provide a nearlyabsolute selection for true targeted events. Southern hybridizationanalysis is confirmatory.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 # 19               GAC                                                        - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 16 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    16                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 # 20               ACTC                                                       __________________________________________________________________________

We claim:
 1. A method of producing a clonal cell strain of homologouslyrecombinant secondary somatic non-imnmortalized cells of vertebrateorigin selected from the group consisting of mammalian secondaryfibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, lymphocytes, bone marrow cells, muscle cells,hepatocytes and precursors thereof, said method comprising the stepsof:a) transfecting a primary or secondary somatic non-imnmortalized cellof vertebrate origin with a DNA construct comprising exogenous DNA whichincludes a DNA sequence homologous to a genomic DNA sequence of theprimary or secondary cell, thereby producing a transfected primary orsecondary cell, and wherein said exogenous DNA further comprises a DNAsequence that encodes a therapeutic product selected from the groupconsisting of enzymes, cytokines, hormones, antigens, antibodies,clotting factors, regulatory proteins, transcription proteins, andreceptors; b) maintaining the transfected primary or secondary cellunder conditions appropriate for homologous recombination between a DNAsequence in the DNA construct and genomic DNA to occur, therebyproducing a homologously recombinant primary or secondary cell; and c)culturing the homologously recombinant primary or secondary cell underconditions appropriate for propagating the homologously recombinantprimary or secondary cell, thereby producing a clonal cell strain ofhomologously recombinant secondary somatic non-imnmortalized cells,wherein the clonal cell strain supplies said therapeutic product.
 2. Themethod of claim 1 wherein the DNA construct of (a) additionally includesDNA encoding a selectable marker.
 3. A clonal cell strain ofhomologously recombinant secondary somatic non-imnmortalized cellsproduced by the method of claim
 1. 4. The method of claim 1, wherein thecells of the clonal cell strain of homologously recombinant secondarysomatic non-immortalized cells of vertebrate origin are differentiated.5. A method of producing a clonal cell strain of homologouslyrecombinant secondary somatic non-immortalized cells of vertebrateorigin, said method comprising the steps of:a) providing a DNA constructcomprising the three elements:1) exogenous DNA encoding a product to beexpressed in a primary or secondary somatic cell of vertebrate origin;2) a DNA sequence homologous with a genomic DNA sequence in the primaryor secondary somatic cell of vertebrate origin; and 3) a DNA sequenceencoding at least one selectable marker; b) transfecting a primary orsecondary somatic cell with the DNA construct provided in (a), therebyproducing a primary or secondary cell containing the DNA construct; c)maintaining the primary or secondary somatic cell produced in (b) underconditions appropriate for homologous recombination to occur betweengenomic DNA and a DNA sequence homologous with genomic DNA, therebyproducing a homologously recombinant primary or secondary cell ofvertebrate origin having the DNA construct of (a) integrated intogenomic DNA of the primary or secondary cell; and d) culturing thehomologously recombinant primary or secondary cell under conditionsappropriate for propagating the homologously recombinant primary orsecondary cell, thereby producing a clonal cell strain of homologouslyrecombinant secondary somatic non-immortalized cells, wherein the clonalcell strain supplies said product.
 6. The method of claim 5 wherein theexogenous DNA is itself a therapeutic product selected from the groupconsisting of: DNA sequences sufficient for sequestration of a proteinor nucleic acid in the cell, DNA sequences which bind to a cellularregulatory protein, DNA sequences which alter secondary or tertiarychromosomal structure and DNA sequences which are transcriptionalregulatory elements.
 7. The method of claim 5 wherein the primary orsecondary somatic cell is selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, lymphocytes, bone marrow cells muscle cells,hepatocytes and precursors thereof.
 8. The method of claim 5 wherein theprimary or secondary somatic cell is of mammalian origin.
 9. The methodof claim 5 wherein the primary or secondary somatic cell is selectedfrom the group consisting of: primary human cells, secondary humancells, primary rabbit cells and secondary rabbit cells.
 10. The methodof claim 5 wherein the exogenous DNA encodes a therapeutic productselected from the group consisting of: enzymes, cytokines, hormones,antigens, antibodies, clotting factors, regulatory proteins,transcription proteins and receptors.
 11. A clonal cell strain ofhomologously recombinant secondary somatic non-immortalized cellsproduced by the method of claim
 5. 12. A method of producing a clonalcell strain of homologously recombinant secondary somaticnon-immortalized cells of vertebrate origin, said method comprising thesteps of:a) providing a DNA construct comprising:1) exogenous DNAsequences encoding a product not normally expressed in a primary orsecondary somatic cell of vertebrate origin, or not expressed insignificant levels in the primary or secondary somatic cell as obtained;2) a DNA sequence homologous with a genomic DNA sequence in the primaryor secondary somatic cell; and 3) a DNA sequence encoding at least oneselectable marker; b) transfecting a primary or secondary somaticnon-immortalized cell with the DNA construct provided in (a), therebyproducing a primary or secondary cell containing the DNA construct; c)maintaining the primary or secondary cell produced in (b) underconditions appropriate for homologous recombination to occur betweengenomic DNA and a DNA sequence homologous with genomic DNA, therebyproducing a homologously recombinant primary or secondary cell ofvertebrate origin having the DNA construct of (a) integrated intogenomic DNA of the primary or secondary cell; and d) culturing thehomologously recombinant primary or secondary cell under conditionsappropriate for propagating the homologously recombinant primary orsecondary cell, thereby producing a clonal cell strain of homologouslyrecombinant secondary somatic non-imnmortalized cells, wherein theclonal cell strain supplies said product.
 13. A method of producing aclonal cell strain of homologously recombinant secondary somaticnon-immortalized cells of vertebrate origin, said method comprising thesteps of:a) providing a DNA construct comprising the three elements:1)exogenous DNA encoding a product to be expressed in a primary orsecondary somatic cell of vertebrate origin; 2) a DNA sequencehomologous with a genomic DNA sequence in the primary or secondarysomatic cell of vertebrate origin; and 3) a DNA sequence encoding atleast one selectable marker; b) transfecting a primary or secondarysomatic non-immortalized cell with the DNA construct provided in (a),thereby producing a primary or secondary cell containing the DNAconstruct; c) maintaining the primary or secondary cell produced in (b)under conditions appropriate for homologous recombination to occurbetween genomic DNA and a DNA sequence homologous with genomic DNA,thereby producing a homologously recombinant primary or secondary cellof vertebrate origin having the DNA construct of (a) integrated intogenomic DNA of the primary or secondary cell; d) exposing thehomologously recombinant primary or secondary cell produced in (c) to aselective agent which selects for cells expressing the selectable markerpresent in the targeting DNA construct, whereby a cell that has stablyintegrated the selectable marker can survive and form colonies; e)screening colonies produced in (d) to identify the homologouslyrecombinant primary or secondary cell; and f) culturing the homologouslyrecombinant primary or secondary cell identified in (e) under conditionsappropriate for propagating the homologously recombinant primary orsecondary cell, thereby producing a clonal cell strain of homologouslyrecombinant secondary somatic non-immortalized cells, wherein the clonalcell strain supplies the product.
 14. The method of claim 13 wherein theselective marker is neo and selective agent is G418.
 15. A method ofproducing a clonal cell strain of homologously recombinant secondarysomatic non-immortalized cells of vertebrate origin, said methodcomprising the steps of:a) providing a DNA construct comprising thethree elements:1) exogenous DNA encoding a product to be expressed in aprimary or secondary somatic cell of vertebrate origin; 2) DNA targetingsequences homologous with genomic DNA sequences in the primary orsecondary somatic cell of vertebrate origin; and 3) DNA sequencesencoding at least one positive selection marker and one negativeselection marker, in such a configuration that homologous recombinationbetween the targeting sequences and homologous sequences in the primaryor secondary somatic cell genome results in the targeted integration ofthe positive selection marker, while the negative selection marker isnot integrated; b) transfecting a primary or secondary somaticnon-immortalized cell with the DNA construct provided in (a), therebyproducing a primary or secondary cell containing the DNA construct; c)maintaining the primary or secondary cell produced in (b) underconditions appropriate for homologous recombination to occur betweengenomic DNA and a DNA sequence homologous with genomic DNA, therebyproducing a homologously recombinant primary or secondary cell ofvertebrate origin having the DNA of (a(1)) integrated into genomic DNAof the primary or secondary cell; d) exposing the primary or secondarycell produced in (c) to a selective agent which selects for cellsexpressing the positive selection marker present in the targeting DNAconstruct, whereby a cell that has not stably integrated the positiveselection marker is killed and a cell that has stably integrated thepositive selection marker survives and forms colonies; e) additionallyexposing the cell produced in (d) to an agent which selects againstcells expressing the negative selection marker present in the targetingDNA construct, whereby a cell that has stably integrated the negativeselection marker is killed, with the result of steps (d) and (e) beingsuch that a homologously recombinant cell is selected; and f) culturingthe homologously recombinant primary or secondary cell under conditionsappropriate for propagating the homologously recombinant primary orsecondary cell, thereby producing a clonal cell strain of homologouslyrecombinant secondary somatic non-immortalized cells, wherein the clonalcell strain supplies the product.
 16. The method of claim 15 wherein theprimary or secondary somatic cell is selected from the group consistingof: fibroblasts, keratinocytes, epithelial cells, endothelial cells,glial cells, neural cells, lymphocytes, bone marrow cells, muscle cells,hepatocytes and precursors thereof.
 17. The method of claim 15 whereinthe primary or secondary somatic cell is of mammalian origin.
 18. Themethod of claim 15 wherein the primary or secondary somatic cell isselected from the group consisting of: primary human cells, secondaryhuman cells, primary rabbit cells and secondary rabbit cells.
 19. Themethod of claim 15 wherein the exogenous DNA encodes a therapeuticproduct selected from the group consisting of: enzymes, cytokines,hormones, antigens, antibodies, clotting factors, regulatory proteins,transcription proteins and receptors.
 20. The method of claim 15 inwhich the positive selective marker is neo and the selective agent isG418.
 21. The method of claim 15 in which the negative selection markeris gpt and the negative selective agent is 6-thioxanthine.
 22. Themethod of claim 15 in which the negative selection marker is HSV-TK geneand the negative selective agent is gancyclovir.
 23. The method of claim15 in which two negative selection markers are used, where one negativeselection marker is gpt and one negative selection marker is HSV-TKgene.
 24. A clonal cell strain of homologously recombinant secondarysomatic non-immortalized cells produced by the method of claim 15.